- Email: [email protected]
The control of puffing by ions: A reply
Molecular and Cellular Endocrinology,
0 Elsevier/North-Holland
I (1977) 105-110 Scientific Publishers, Ltd.
THE CONTROL OF PUFFING
BY IONS: A REPLY
Heinrich KROEGER Institut fiir Genetik, Universitli’t des Saarlandes, 6600 Saarbriicken,
G.F.R.
Received 11 January 1977
In a ‘Critical Review’ Ashburner and Cherbas (1976) reexamine the evidence that has been presented with regard to the suggestion that the cell’s electrolytes are a determinant of its pattern of gene activities. They conclude that they cannot exclude this model but see little reason to believe in it. In the present reply I argue that they use inappropriate computation procedures, oversimplify essential aspects of cellular electrolyte metabolism and make unfounded assumptions as to what has actually been proposed. In addition I show that they abstract the literature incorrectly. I maintain that the negative conclusions reached by Ashburner and Cherbas are unfounded and that the issue remains open. Keywords:
chromosome puffing; Kroeger hypothesis; Na+; K+; ecdysone; insects.
What Ashburner and Cherbas in their ‘Critical Review’ (1976; henceforth A & C) refer to as the ‘Kroeger hypothesis’ is the suggestion that chromosomal sites become active and inactive during biological events such as developmental progress, the cell cycle, the impact of certain hormones, etc., by responding to controlled shifts in the electrolyte milieu of the nucleus, i.e. that the electrolyte status of a cell is a major determinant of its genomic activity pattern. This proposition is based on two premises: (a) the occurrence of controlled electrolyte shifts, notably of Na+, K+ and Mg2+, during the above events, and (b) the discovery of a subtle differential sensitivity of chromosomal sites towards such shifts. Both these premises seem now well substantiated. It is true that A & C, with regard to the differential sensitivity of chromosomal sites, question “that sufficient specificity has actually been demonstrated” (p. 105), but they conclude themselves that certain sites are “differentially sensitive to cations under some conditions” (p. 105). Further testing of our suggestion thus seems to hinge essentially on a quantitative argument: can it be shown that the natural variation of cellular electrolytes occurs within the range in which chromosomal sites respond, i.e. within their sensitivity range? If one employs semiquantitative computation procedures, it is possible to demonstrate that this is the case (see below, I(a)). However, a full quantitation of the relationship between every single chromosomal site, its puffing program and the 105
106
H. Kroeger
variation of single components of the cell’s electrolyte milieu is still distant. In such a situation a thorough evaluation of the available evidence by outside critics must be welcome, because there is always the danger that proponents of a hypothesis view supporting evidence out of proportion and miss essential counterarguments and control experiments. Unfortunately, the ‘Critical Review’ presented by A & C is, nearly paragraph by paragraph, beset with oversimplifications concerning the electrolyte metabolism of cells, unsound computation procedures and misconceptions as to what has been actually proposed. In addition it is sown with incorrect abstractions of the published literature. To lay open all these defects would require an inordinate amount of space and would frustrate the reader. I will therefore present selected examples only and in doing so, I will direct my efforts towards a clarification of points which have in part not been fully discussed in the literature and thus may indeed be confusing to the reader.
I. UNSOUND COMPUTATIONS
AND MISCONCEPTIONS
(a) Computation of inductive jields relative to intracellular ion concentrations (IFi 5) In principle it is possible to compute those ranges of intracellular Na+/K+ concentrations which induce puffing at a particular locus (called ‘inductive fields relative to intracellular ion concentrations’ (IF’%) in a two-dimensional Na+/K+ plot) from (a) data on the respective extracellular ranges (IFe’s) (Kroeger and Mtiller, 1973) and (b) [Na+le: [Na+]’ and [K+le: [K+]’ ratios as determined by Kroeger and Trosch (1974). However, we have so far refrained from a detailed computation of IF’% because we feel that certain aspects need to be further elucidated. To give a preliminary conception, Kroeger et al. (1973) in their fig. 4 present estimates of IF’ borderlines and their relation to those ion shifts which occur during development. Their comparison shows clearly that the approximate ranges of nuclear ion concentrations and their shifts overlap with the regions of ion sensitivities of chromosomal loci, thus providing, although as yet only on a semiquantitative basis, exactly that piece of evidence on which, as stated above, the acceptability of our suggestion seems to hinge. A & C also attempt such a comparison but unfortunately they employ an entirely inappropriate procedure to derive IFi’s. Without taking recourse to the regression parameters provided by Kroeger and Trijsch (1974) which allow for a point-bypoint transformation of IFe’s into IFi’s, and without an effort either to subtract the measuring error or to consider the contribution of ‘nuclei with inverted [Nat]‘/ [K+]’ ratios’ (see Kroeger et al., 1973) they form the smallest convex polygon which encloses all [Na+]‘; [K+]’ determinations from a particular IFe and consider this, although with restrictions, as an IF’. Obviously the scatter inherent in the [Na+li; [K+]’ determinations must displace the boundaries from their true position
and must do so towards an e~~~~~~~e~~of the polygons. This procedure amounts to selecting the IrigMt value measured in a series of determinations as representative instead of its x - one single ‘off measurement in one polygon will make comparisons between IF’% pointless. Any conclusions A & C derive from the use of this procedure are therefore valueless. (6) Ion specificity vs. osmolarity
Exposure of cells to anisotonic media has a double effect on their cation content: (1) swift wafer flmes out of the cell (or into it) concentrate (or dilute) all molecules in the cell’s solute, among these of course Na+ and K+; (2) superimposed are slower effects produced by the permeation of these cations through the cell’s membranes. Both vary [Na]‘; [Kli, but the former effect, which is purely dependent on the osmoIarity of the medium, should be the more precise and reliable one, because the permeation of cations through membranes is slower and is subject to a number of complicating factors such as permeability characteristics, active transport and problems of anion permeability. Unfortunately A & C omit all such considerations, take osmotic pressure and intracellular effects of specific-ions ~rou~out their paper as u~r~~t~~e~ and, stating that the osmolarity of a medium seems to be the better predictor in the control of puffing than its cation composition, conclude that it is not [Na”Ji; [K+]’ which is operative in the control of puffing. (cl Ion species operative in genomic control We suggest that the nuclear ionic milieu, by variation of its components, controls the puffing activity of chrot~osomal sites. However, we cannot possibly predict waist ion species are varied by the cell at w~ut instances to effect a switchover of its genomic activity pattern. It seems plausible that Na’, Kf and Mg2’ are preponderant at least in certain phases of development, simply because the sites in question respond particularly well to their variation. However, that other ionic species are also involved in the controlling function has been expected from the earliest days of the suggestion; it has experimentally been supported for amino acids (Gopalan, 1973; Gopalan and Robert, 1976). Therefore, instances where the [Na’]‘; [K’]’ component does not suffice to explain the installation of a puffing pattern must not be taken to contradict our proposition. What can be deduced from the joint effects of Naf, Kf and Mg” and the way they add up to influence chromosomes loci is that the effective ~~~~~i~ is the to&l ionic milrieu, while the specificity of the effects depends on the relative concentrations of its components. Thus a locus classed as ‘K’ sensitive’ does not, as A & C seem to assume throu~out their Review, respond solely to [K’]. Instead it will react with a somewhat higher sensitivity to a shift in [K’] than to other parameters tested so far. The effectivity of any ion shifts will always depend on the whole remainder of components in the electrolyte milieu, the ‘ionic background’ (see Lezzi and Robert, 1972). It is thus not permissible to compare Na*/K+ sensitivities
H. Kroeger
108
as determined in the presence of the ‘background’ ion milieu (i.e. in situ), with those determined in their absence, i.e. in pure NaCl/KCl solutions or similar media.
II. INCORRECT
ABSTRACTION
OF LITERATURE
(a) Ion sensitivities in isolated chromosomes or nuclei The most striking evidence on the existence and range of differential ion sensitivities comes from puffing studies of isolated nuclei or polytene chromosomes exposed to media which contain nothing but the ion species to be tested in varying proportions (Lezzi, 1966; Lezzi and Gilbert, 1970; Robert, 1971). The differential reactivity of chromosomal sites thus demonstrated constitutes evidence of a directness and lucidity very seldom encountered in biological studies and the reactivity is on the whole exactly of the kind predicted by us. A & C do regard these results as “impressive” (p. 104), but despite this they “cannot view these data as strong evidence in favour of Kroeger’s hypothesis . .” (p. 104). They give three reasons: (1) The concentrations tested are not reasonable intranuclear concentrations. Obviously A & C have not carefully read the ‘Methods’ section in the relevant paper; Lezzi (1966, p. 572) states clearly that the Na+/K+ amounts which A&C discuss actually contribute only l/4 to the final volume. If one corrects their numerical data using the ion contents of nuclei (Kroeger et al., 1973), one finds the resulting concentrations to be “reasonable” and they do, contrary to what A & C consider, fall into the “normal range” of [Na+li; [K+]‘; they thus constitute exactly the experiment A & C feel “is required” (p. 104). Sensitivities from the other paper cited by A & C (Lezzi and Gilbert, 1970) must not be directly compared because they have been studied in the absence of the ‘ionic background’ (see I(c)). (2) A & C find that “the ionic concentrations effective for nuclei and isolated chromosomes do not fall within the relevant IF’s” (p. 105) - but it is their (in part wrongly computed and in part not comparable, see above) concentrations which do not fall into their (unsoundly computed, see I(a)) IFi’s. (3) A & C remain unconvinced that sufficient specificity has actually been demonstrated, because “one reason may be that many regions faded in K+ solutions and many in Na+ solutions” (p. 105). - If one consults the original paper by Lezzi and Gilbert (1970) one finds that these authors never say that “many” bands faded; instead they state that “several” bands do; at another place they state even more clearly that “single” bands fade. This misquotation is crucial. If, of the about 2000 bands present in the nucleus, ‘many’ faded, one may argue that any two may be ‘by chance’ among these. If, however, “single” ones or just “several” out of this large number fade, as is actually stated by the original authors, the chance argument collapses. That indeed more bands respond than just I-18-C and I-19-A must be expected because a number of other puffs make their appearance concomitant with these, probably responding to the same stimulus. In addition one would expect
109
The control of puffing by ions: a reply
more bands to react because the Na+; K+ concentrations and combinations tested over a more extreme range than occurs during development. (b) Change in potential difference
were
(PO)
A & C write (on p. 102): “Kroeger (1966) published experiments showing that ecdysone, when given to explanted glands in culture, may cause an increase (of 35 mV) in the potential difference across the plasma membrane.” They cite a paper by Hax et al. (1974) as not supporting the hypothesis, because, as A & C state, “No effects were found on the cell’s membrane potential nor were any consistent effects found on the resistances of either the junctional or nonjunctional cell membranes.” If one consults Kroeger’s (1966) original paper one finds that the average increase in the PD was about 12, not 35 mV, and if one consults the paper by Hax et al. (1974) one reads that upon exposure to ecdysterone “A straight increase as well as an initial decrease in junctional permeability was observed”and “Again the parallelism, although less pronounced, between the hyperpolarization of the outer membrane and the increase in communication seems to be present.” This reveals that A & C have abstracted the exact opposite of what the original authors report. (c) Omissions
It is the privilege of reviewers to select from the literature what they consider pertinent and to omit other pieces of evidence. It does not seem permissible, however, to leave out in such a review the following pieces of supporting evidence: (1) the fact that in C/z. tentans two more regions (IV 2 B. BRl) were found to respond to K+ and Naf (Lezzi, 1967); (2) a demonstration of similar ion specificities for homologous bands in Ch. thummi (Robert, 1971), along with two more regions which exhibit a differential Na+/K+ sensitivity; (3) all the evidence relating to Mg*+ sensitive loci (Lezzi, 1967; Lezzi and Robert, 1972); and (4) the whole question of juvenile hormone inducibility of certain puffs in accord with such predictions (Lezzi and Frigg, 1971; Lezzi, 1974). The experiment that A & C regret never to their knowledge to have been done is described in a paper by Korochkina et al. (1972), an English translation of which was kindly offered to me by Ashburner in 1974.
REFERENCES Ashburncr, M. and Cherbas, P. (1976) Mol. Cell. Endocrinol. 5,89-107. Gopalan, H. (1973) Chromosoma 44, 25-42. Gopalan, H. and Robert, M. (1976) J. Cell Biol. 70,418a. Hax, W.M.A., Venrooij, G. and Vossenberg, J. (1974) J. Membrane Biol. 19,253-266. Korochkina, L.S.,Kiknadze, 1.1. and Muradov, S.V. (1972) Ontogenez 3, 177-186. Kroeger, H. (1966) Exp. Cell Res. 41,64-80. Kroeger, H. and Miillcr, G. (1973) Exp. Ccl1 Res. 82,89-94. Krocgcr, H. and TrBsch, W. (1974) J. Cell. Physiol. 83, .19-26.
110
H. Kroeger
Kroeger, H., Trosch, W. and Miiller, G. (1973) Exp. Cell Res. 80, 329-339. Lezzi, M. (1966) Exp. Cell Res. 43,571-577. Lezzi, M. (1967) Chromosoma 21, 109-122. Lezzi, M. (1974) Mol. Cell. Endocrinol. 1, 189-207, Lezzi, M. and Frigg, M. (1971) Mitt. Schweiz. Entomol. Ges. 44, 163-170. Lezzi, M. and Gilbert, L.I. (1970) J. Cell Sci. 6,615-627. Lezzi, M. and Robert, M. (1972) In: Developmental Studies on Giant Chromosomes, Beermann (Springer, Berlin). Robert, M. (1971) Chromosoma 36, l-33.
Ed.: W.
Comment from the Managing Editor. Drs. Ashburner and Cherbas have read Professor Kroeger’s comments. They remain unconvinced and stand by the views they expressed in their paper. They feel that both sides of this controversy have been adequately represented and that it now remains for readers to form their own opinions.
0 Elsevier/North-Holland
I (1977) 105-110 Scientific Publishers, Ltd.
THE CONTROL OF PUFFING
BY IONS: A REPLY
Heinrich KROEGER Institut fiir Genetik, Universitli’t des Saarlandes, 6600 Saarbriicken,
G.F.R.
Received 11 January 1977
In a ‘Critical Review’ Ashburner and Cherbas (1976) reexamine the evidence that has been presented with regard to the suggestion that the cell’s electrolytes are a determinant of its pattern of gene activities. They conclude that they cannot exclude this model but see little reason to believe in it. In the present reply I argue that they use inappropriate computation procedures, oversimplify essential aspects of cellular electrolyte metabolism and make unfounded assumptions as to what has actually been proposed. In addition I show that they abstract the literature incorrectly. I maintain that the negative conclusions reached by Ashburner and Cherbas are unfounded and that the issue remains open. Keywords:
chromosome puffing; Kroeger hypothesis; Na+; K+; ecdysone; insects.
What Ashburner and Cherbas in their ‘Critical Review’ (1976; henceforth A & C) refer to as the ‘Kroeger hypothesis’ is the suggestion that chromosomal sites become active and inactive during biological events such as developmental progress, the cell cycle, the impact of certain hormones, etc., by responding to controlled shifts in the electrolyte milieu of the nucleus, i.e. that the electrolyte status of a cell is a major determinant of its genomic activity pattern. This proposition is based on two premises: (a) the occurrence of controlled electrolyte shifts, notably of Na+, K+ and Mg2+, during the above events, and (b) the discovery of a subtle differential sensitivity of chromosomal sites towards such shifts. Both these premises seem now well substantiated. It is true that A & C, with regard to the differential sensitivity of chromosomal sites, question “that sufficient specificity has actually been demonstrated” (p. 105), but they conclude themselves that certain sites are “differentially sensitive to cations under some conditions” (p. 105). Further testing of our suggestion thus seems to hinge essentially on a quantitative argument: can it be shown that the natural variation of cellular electrolytes occurs within the range in which chromosomal sites respond, i.e. within their sensitivity range? If one employs semiquantitative computation procedures, it is possible to demonstrate that this is the case (see below, I(a)). However, a full quantitation of the relationship between every single chromosomal site, its puffing program and the 105
106
H. Kroeger
variation of single components of the cell’s electrolyte milieu is still distant. In such a situation a thorough evaluation of the available evidence by outside critics must be welcome, because there is always the danger that proponents of a hypothesis view supporting evidence out of proportion and miss essential counterarguments and control experiments. Unfortunately, the ‘Critical Review’ presented by A & C is, nearly paragraph by paragraph, beset with oversimplifications concerning the electrolyte metabolism of cells, unsound computation procedures and misconceptions as to what has been actually proposed. In addition it is sown with incorrect abstractions of the published literature. To lay open all these defects would require an inordinate amount of space and would frustrate the reader. I will therefore present selected examples only and in doing so, I will direct my efforts towards a clarification of points which have in part not been fully discussed in the literature and thus may indeed be confusing to the reader.
I. UNSOUND COMPUTATIONS
AND MISCONCEPTIONS
(a) Computation of inductive jields relative to intracellular ion concentrations (IFi 5) In principle it is possible to compute those ranges of intracellular Na+/K+ concentrations which induce puffing at a particular locus (called ‘inductive fields relative to intracellular ion concentrations’ (IF’%) in a two-dimensional Na+/K+ plot) from (a) data on the respective extracellular ranges (IFe’s) (Kroeger and Mtiller, 1973) and (b) [Na+le: [Na+]’ and [K+le: [K+]’ ratios as determined by Kroeger and Trosch (1974). However, we have so far refrained from a detailed computation of IF’% because we feel that certain aspects need to be further elucidated. To give a preliminary conception, Kroeger et al. (1973) in their fig. 4 present estimates of IF’ borderlines and their relation to those ion shifts which occur during development. Their comparison shows clearly that the approximate ranges of nuclear ion concentrations and their shifts overlap with the regions of ion sensitivities of chromosomal loci, thus providing, although as yet only on a semiquantitative basis, exactly that piece of evidence on which, as stated above, the acceptability of our suggestion seems to hinge. A & C also attempt such a comparison but unfortunately they employ an entirely inappropriate procedure to derive IFi’s. Without taking recourse to the regression parameters provided by Kroeger and Trijsch (1974) which allow for a point-bypoint transformation of IFe’s into IFi’s, and without an effort either to subtract the measuring error or to consider the contribution of ‘nuclei with inverted [Nat]‘/ [K+]’ ratios’ (see Kroeger et al., 1973) they form the smallest convex polygon which encloses all [Na+]‘; [K+]’ determinations from a particular IFe and consider this, although with restrictions, as an IF’. Obviously the scatter inherent in the [Na+li; [K+]’ determinations must displace the boundaries from their true position
and must do so towards an e~~~~~~~e~~of the polygons. This procedure amounts to selecting the IrigMt value measured in a series of determinations as representative instead of its x - one single ‘off measurement in one polygon will make comparisons between IF’% pointless. Any conclusions A & C derive from the use of this procedure are therefore valueless. (6) Ion specificity vs. osmolarity
Exposure of cells to anisotonic media has a double effect on their cation content: (1) swift wafer flmes out of the cell (or into it) concentrate (or dilute) all molecules in the cell’s solute, among these of course Na+ and K+; (2) superimposed are slower effects produced by the permeation of these cations through the cell’s membranes. Both vary [Na]‘; [Kli, but the former effect, which is purely dependent on the osmoIarity of the medium, should be the more precise and reliable one, because the permeation of cations through membranes is slower and is subject to a number of complicating factors such as permeability characteristics, active transport and problems of anion permeability. Unfortunately A & C omit all such considerations, take osmotic pressure and intracellular effects of specific-ions ~rou~out their paper as u~r~~t~~e~ and, stating that the osmolarity of a medium seems to be the better predictor in the control of puffing than its cation composition, conclude that it is not [Na”Ji; [K+]’ which is operative in the control of puffing. (cl Ion species operative in genomic control We suggest that the nuclear ionic milieu, by variation of its components, controls the puffing activity of chrot~osomal sites. However, we cannot possibly predict waist ion species are varied by the cell at w~ut instances to effect a switchover of its genomic activity pattern. It seems plausible that Na’, Kf and Mg2’ are preponderant at least in certain phases of development, simply because the sites in question respond particularly well to their variation. However, that other ionic species are also involved in the controlling function has been expected from the earliest days of the suggestion; it has experimentally been supported for amino acids (Gopalan, 1973; Gopalan and Robert, 1976). Therefore, instances where the [Na’]‘; [K’]’ component does not suffice to explain the installation of a puffing pattern must not be taken to contradict our proposition. What can be deduced from the joint effects of Naf, Kf and Mg” and the way they add up to influence chromosomes loci is that the effective ~~~~~i~ is the to&l ionic milrieu, while the specificity of the effects depends on the relative concentrations of its components. Thus a locus classed as ‘K’ sensitive’ does not, as A & C seem to assume throu~out their Review, respond solely to [K’]. Instead it will react with a somewhat higher sensitivity to a shift in [K’] than to other parameters tested so far. The effectivity of any ion shifts will always depend on the whole remainder of components in the electrolyte milieu, the ‘ionic background’ (see Lezzi and Robert, 1972). It is thus not permissible to compare Na*/K+ sensitivities
H. Kroeger
108
as determined in the presence of the ‘background’ ion milieu (i.e. in situ), with those determined in their absence, i.e. in pure NaCl/KCl solutions or similar media.
II. INCORRECT
ABSTRACTION
OF LITERATURE
(a) Ion sensitivities in isolated chromosomes or nuclei The most striking evidence on the existence and range of differential ion sensitivities comes from puffing studies of isolated nuclei or polytene chromosomes exposed to media which contain nothing but the ion species to be tested in varying proportions (Lezzi, 1966; Lezzi and Gilbert, 1970; Robert, 1971). The differential reactivity of chromosomal sites thus demonstrated constitutes evidence of a directness and lucidity very seldom encountered in biological studies and the reactivity is on the whole exactly of the kind predicted by us. A & C do regard these results as “impressive” (p. 104), but despite this they “cannot view these data as strong evidence in favour of Kroeger’s hypothesis . .” (p. 104). They give three reasons: (1) The concentrations tested are not reasonable intranuclear concentrations. Obviously A & C have not carefully read the ‘Methods’ section in the relevant paper; Lezzi (1966, p. 572) states clearly that the Na+/K+ amounts which A&C discuss actually contribute only l/4 to the final volume. If one corrects their numerical data using the ion contents of nuclei (Kroeger et al., 1973), one finds the resulting concentrations to be “reasonable” and they do, contrary to what A & C consider, fall into the “normal range” of [Na+li; [K+]‘; they thus constitute exactly the experiment A & C feel “is required” (p. 104). Sensitivities from the other paper cited by A & C (Lezzi and Gilbert, 1970) must not be directly compared because they have been studied in the absence of the ‘ionic background’ (see I(c)). (2) A & C find that “the ionic concentrations effective for nuclei and isolated chromosomes do not fall within the relevant IF’s” (p. 105) - but it is their (in part wrongly computed and in part not comparable, see above) concentrations which do not fall into their (unsoundly computed, see I(a)) IFi’s. (3) A & C remain unconvinced that sufficient specificity has actually been demonstrated, because “one reason may be that many regions faded in K+ solutions and many in Na+ solutions” (p. 105). - If one consults the original paper by Lezzi and Gilbert (1970) one finds that these authors never say that “many” bands faded; instead they state that “several” bands do; at another place they state even more clearly that “single” bands fade. This misquotation is crucial. If, of the about 2000 bands present in the nucleus, ‘many’ faded, one may argue that any two may be ‘by chance’ among these. If, however, “single” ones or just “several” out of this large number fade, as is actually stated by the original authors, the chance argument collapses. That indeed more bands respond than just I-18-C and I-19-A must be expected because a number of other puffs make their appearance concomitant with these, probably responding to the same stimulus. In addition one would expect
109
The control of puffing by ions: a reply
more bands to react because the Na+; K+ concentrations and combinations tested over a more extreme range than occurs during development. (b) Change in potential difference
were
(PO)
A & C write (on p. 102): “Kroeger (1966) published experiments showing that ecdysone, when given to explanted glands in culture, may cause an increase (of 35 mV) in the potential difference across the plasma membrane.” They cite a paper by Hax et al. (1974) as not supporting the hypothesis, because, as A & C state, “No effects were found on the cell’s membrane potential nor were any consistent effects found on the resistances of either the junctional or nonjunctional cell membranes.” If one consults Kroeger’s (1966) original paper one finds that the average increase in the PD was about 12, not 35 mV, and if one consults the paper by Hax et al. (1974) one reads that upon exposure to ecdysterone “A straight increase as well as an initial decrease in junctional permeability was observed”and “Again the parallelism, although less pronounced, between the hyperpolarization of the outer membrane and the increase in communication seems to be present.” This reveals that A & C have abstracted the exact opposite of what the original authors report. (c) Omissions
It is the privilege of reviewers to select from the literature what they consider pertinent and to omit other pieces of evidence. It does not seem permissible, however, to leave out in such a review the following pieces of supporting evidence: (1) the fact that in C/z. tentans two more regions (IV 2 B. BRl) were found to respond to K+ and Naf (Lezzi, 1967); (2) a demonstration of similar ion specificities for homologous bands in Ch. thummi (Robert, 1971), along with two more regions which exhibit a differential Na+/K+ sensitivity; (3) all the evidence relating to Mg*+ sensitive loci (Lezzi, 1967; Lezzi and Robert, 1972); and (4) the whole question of juvenile hormone inducibility of certain puffs in accord with such predictions (Lezzi and Frigg, 1971; Lezzi, 1974). The experiment that A & C regret never to their knowledge to have been done is described in a paper by Korochkina et al. (1972), an English translation of which was kindly offered to me by Ashburner in 1974.
REFERENCES Ashburncr, M. and Cherbas, P. (1976) Mol. Cell. Endocrinol. 5,89-107. Gopalan, H. (1973) Chromosoma 44, 25-42. Gopalan, H. and Robert, M. (1976) J. Cell Biol. 70,418a. Hax, W.M.A., Venrooij, G. and Vossenberg, J. (1974) J. Membrane Biol. 19,253-266. Korochkina, L.S.,Kiknadze, 1.1. and Muradov, S.V. (1972) Ontogenez 3, 177-186. Kroeger, H. (1966) Exp. Cell Res. 41,64-80. Kroeger, H. and Miillcr, G. (1973) Exp. Ccl1 Res. 82,89-94. Krocgcr, H. and TrBsch, W. (1974) J. Cell. Physiol. 83, .19-26.
110
H. Kroeger
Kroeger, H., Trosch, W. and Miiller, G. (1973) Exp. Cell Res. 80, 329-339. Lezzi, M. (1966) Exp. Cell Res. 43,571-577. Lezzi, M. (1967) Chromosoma 21, 109-122. Lezzi, M. (1974) Mol. Cell. Endocrinol. 1, 189-207, Lezzi, M. and Frigg, M. (1971) Mitt. Schweiz. Entomol. Ges. 44, 163-170. Lezzi, M. and Gilbert, L.I. (1970) J. Cell Sci. 6,615-627. Lezzi, M. and Robert, M. (1972) In: Developmental Studies on Giant Chromosomes, Beermann (Springer, Berlin). Robert, M. (1971) Chromosoma 36, l-33.
Ed.: W.
Comment from the Managing Editor. Drs. Ashburner and Cherbas have read Professor Kroeger’s comments. They remain unconvinced and stand by the views they expressed in their paper. They feel that both sides of this controversy have been adequately represented and that it now remains for readers to form their own opinions.